Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
The chloroplasts of higher plants accumulate individual components of the photosynthetic machinery as a relatively large fraction of total cellular protein. The best example is the enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) involved in CO2 fixation which can make up 65% of the total leaf protein (Ellis, R. J. 1979). Because of the potentially attainable high protein levels, there is significant interest in exploring chloroplasts as an alternative system for protein expression. To date, protein levels expressed from transgenes in chloroplasts are below the levels of highly-expressed chloroplast genes. Highest levels reported thus far in leaves are as follows: 1% of neomycin phophotransferase (Carrer et al., 1993); 2.5% β-glucuronidase (Staub and Maliga, 1993) and 3-5% of Bacillus thuringiensis (Bt) crystal toxins (McBride et al., 1995). An alternative system, based on a nuclear-encoded, plastid-targeted T7 RNA polymerase may offer higher levels of protein expression (McBride t al., 1994), although this yield may come at a price.
In bacteria, the rate limiting step of protein synthesis is usually the initiation of translation, involving the binding of the initiator tRNA (formyl-methionyl-tRNAf) and mRNA to the 70S ribosome, recognition of the initiator codon, and the precise phasing of the reading frame of the mRNA. Translation initiation depends on three initiation factors (IF1, IF2, IF3) and requires GTP. The 30S subunit is guided to the initiation codon by RNA—RNA base pairing between the 3′ of the 16S rRNA and the mRNA ribosome binding site, or Shine-Dalgarno (SD) sequence, located about 10 nucleotides upstream of the translation initiation codon (Voorma, 1996). RNA—RNA interaction between the “downstream box” (DB), a 15 nt sequence downstream of the AUG translational initiation codon and complementary sequences in the 16S rRNA 3′ sequence or anti-downstream box (ADB; nucleotide positions 1469-1483) may also facilitate loading of the mRNA onto the 30S ribosome subunit (Sprengart et al., 1996). In addition, specific protein-RNA interactions may also facilitate translation initiation (Voorma, 1996).
Key components of the prokaryotic translation machinery have been identified in plastids, including homologues of the bacterial IF1, IF2 and IF3 initiation factors and an S1-like ribosomal protein (Stern et al., 1997). Most plastid mRNAs (92%) contain a ribosome binding site or SD sequence: GGAGG, or its truncated tri- or tetranucleotide variant. This sequence is similar to the bacterial SD consensus 5′-UAAGGAGGUGA-3′ (SEQ ID NO: 28; Voorma, 1996). High level expression of foreign genes of interest in the plastids of higher plants is extremely desirable. The present invention provides novel genetic translational control elements for use in plastid transformation vectors. Incorporation of these elements into such vectors results in protein expression levels comparable to those observed for highly expressed chloroplast genes in both monocots and dicots.